k*******c
发帖数: 142 | 1 最近构建一个porin-gfp fusion: porin is an out membrane protein; so, the
porin-gfp is not supposed to be fluorescent when they are transported. 是不
是这样呢？
如果用luciferase 是不是好些呢？
多谢 |
S*****s
发帖数: 287 | 2 不应该。有很多文章用 GFP 标记膜蛋白。不过用 GFP 标记蛋白合成出来之后就有信号
，很多荧光信号在 ER 里面。我更喜欢用 HA 或者 FLAG tag 标记蛋白，然后用荧光标
记的抗体做染色，这样能看膜表达也能看全细胞表达。要是你用 GFP 标记蛋白可以直
接做 live cell imaging，看你具体要做什么实验了。
【在 k*******c 的大作中提到】
: 最近构建一个porin-gfp fusion: porin is an out membrane protein; so, the
: porin-gfp is not supposed to be fluorescent when they are transported. 是不
: 是这样呢？
: 如果用luciferase 是不是好些呢？
: 多谢
|
k*******c
发帖数: 142 | 3 thanks for your replying.
we are using bacteria. what I found here:
Green fluorescent protein (GFP) is an ideal cytoplasmic marker because it
fluoresces only when located in the cytoplasm (19, 20). When GFP is targeted
to the periplasm through fusion with a membrane-spanning domain (MSD), it
fails to fold properly and does not fluoresce.
what do you think?
thanks again |
a*****y
发帖数: 277 | 4 You are right. When GFP is in the periplasm it is not folded properly.
However some outer membrane, when expressed recombinantly, can be in both
inner membrane and outer membrane, in which case you'll see GFP Fluorescence
.
There are certainly other fluorescent proteins - I can not remember the name
now. it is called something like ilov or ilove or ilike...? |
s******y
发帖数: 28562 | 5 有专门为膜蛋白设计的GFP variant, 比方说Us9-GFP
而且即使是一般的GFP, 如果把linker设计得长一些，然后把荧光蛋白放在C terminus,
一般还是能有信号的。
Luciferase 不适合作为一般的光学成像，最好不要用。
【在 k*******c 的大作中提到】
: 最近构建一个porin-gfp fusion: porin is an out membrane protein; so, the
: porin-gfp is not supposed to be fluorescent when they are transported. 是不
: 是这样呢？
: 如果用luciferase 是不是好些呢？
: 多谢
|
n***e
发帖数: 180 | 6 wow, 艳阳天MM露面了
terminus,
【在 s******y 的大作中提到】
: 有专门为膜蛋白设计的GFP variant, 比方说Us9-GFP
: 而且即使是一般的GFP, 如果把linker设计得长一些，然后把荧光蛋白放在C terminus,
: 一般还是能有信号的。
: Luciferase 不适合作为一般的光学成像，最好不要用。
|
l*****a
发帖数: 1431 | 7 I didn't get your point here. why HA or Flag tag is better than GFP tag?
Anti-HA or Flag can still give ER staining, if the tagged proteins are
processed in ER. Sorry can't type Chinese in the lab.
【在 S*****s 的大作中提到】
: 不应该。有很多文章用 GFP 标记膜蛋白。不过用 GFP 标记蛋白合成出来之后就有信号
: ，很多荧光信号在 ER 里面。我更喜欢用 HA 或者 FLAG tag 标记蛋白，然后用荧光标
: 记的抗体做染色，这样能看膜表达也能看全细胞表达。要是你用 GFP 标记蛋白可以直
: 接做 live cell imaging，看你具体要做什么实验了。
|
S*****s
发帖数: 287 | 8 I think it is project dependent, and probably investigator dependent, so it
is my personal opinion here. In mammalian cells, GFP shows up as soon as it
is translated, so it can be hard to tell membrane expression from
cytoplasmic expression. In my study, the wild type protein had efficient
membrane trafficking, and mutant protein decreased trafficking efficiency.
GFP fusion protein was not able to show the weak membrane expression of the
mutant protein. When I used HA-tagged protein, I detected membrane
expression by staining UNpermeablized cells, or detected whole cell
expression by staining permeablized cells. GFP fusion protein fluorescence
signal was similar to permeablized cells even if I did not permeablize cells.
I am not fond of GFP also because of its size. I am studying ~55 KDa
membrane protein. GFP protein is ~30KDa. Compared to epitope tags it is too
bulky for my study. But I use it for live cell imaging. It is great to
visualize the GFP fusion protein membrane trafficking in live cells by
monitoring GFP signal after drug treatment.
【在 l*****a 的大作中提到】
: I didn't get your point here. why HA or Flag tag is better than GFP tag?
: Anti-HA or Flag can still give ER staining, if the tagged proteins are
: processed in ER. Sorry can't type Chinese in the lab.
|
